Wenyang-Yiqi Granule Suppresses Oxygen-Glucose Deprivation-Induced Cardiomyocyte Autophagy Through Mammalian Target of Rapamycin Activation in H9c2 Cells

Shuibo Gao, Xiaofang Yu, Lihua Han, Hong Wu
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Abstract

Abstract Background  Wenyang-Yiqi Granule (WYYQ) is a four-component herbal formula, widely used to treat heart failure in China. It is known to regulate autophagy, but the mechanism(s) are unknown. Methods  H9c2 cells were treated with WYYQ for 24 hours prior to oxygen-glucose deprivation (OGD). Expressions of the autophagy markers Beclin-1 and light chain 3 (LC3) were evaluated via quantitative polymerase chain reaction analysis. Protein levels of Beclin-1, LC3, p62, and mammalian targets of rapamycin (mTOR) were determined by Western blot analysis. Transmission electron microscopy was used to explore the effects of WYYQ on autophagosome formation. Results  Treatment with WYYQ dramatically restrained OGD-induced autophagy, which was characterized by an inhibition of Beclin-1 and increased LC3 mRNA expression. In addition, WYYQ decreased the expression of Beclin-1 and the ratio of LC3-II/LC3-I; however, the abundance of p62 was enhanced at the protein level. Manipulation of the LC3-II/LC3-I ratio, p62 abundance, and autophagosome formation in response to WYYQ were associated with mTOR activity. Conclusions  These findings show that WYYQ plays a protective role during hypoxic-ischemic stress through the suppression of excessive autophagy, which may be partially explained by its effects on mTOR. These data provide novel insight into the cardioprotective effects of WYYQ during cardiomyocyte autophagy.
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温阳益气颗粒通过雷帕霉素激活H9c2细胞抑制氧葡萄糖剥夺诱导的心肌细胞自噬
摘要背景温阳益气颗粒是一种四组份的中药方剂,在中国被广泛用于治疗心力衰竭。众所周知,它可以调节自噬,但其机制尚不清楚。方法采用WYYQ处理H9c2细胞24h后进行氧糖剥夺(OGD)。采用定量聚合酶链反应法检测自噬标志物Beclin-1和轻链3 (LC3)的表达。Western blot检测Beclin-1、LC3、p62和哺乳动物雷帕霉素靶蛋白(mTOR)的表达水平。透射电镜观察WYYQ对自噬体形成的影响。结果WYYQ显著抑制ogd诱导的自噬,其特征是抑制Beclin-1,增加LC3 mRNA的表达。此外,WYYQ降低Beclin-1的表达和LC3-II/LC3-I的比值;然而,p62的丰度在蛋白水平上增强。WYYQ对LC3-II/LC3-I比值、p62丰度和自噬体形成的影响与mTOR活性有关。结论WYYQ通过抑制过度自噬在缺氧缺血应激中发挥保护作用,其对mTOR的影响可能是其中的部分原因。这些数据为心肌细胞自噬过程中WYYQ的心脏保护作用提供了新的见解。
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